Objective To observe the abnormal expression of alpha;A-crystallin protein in neural retina in type 2 diabetic rats via proteomic technique.Methods Twenty-eight male Sprague-Dawley (SD) rats were randomly divided into the normal control and the diabetic experimental groups with 14 rats in each group.A type 2 diabetes rat model (T2DM) was set up in the diabetic experimental group by feeding high fat diet combined with peritoneal injection of low dose streptozotocin (STZ);the successful diabetes model is with the randomlydetected blood glucose of ＞16.7 mmol/L.The rats in the control group underwent peritoneal injection of equivalent sodium citrate solution and were fed with normal diet.All of the animals were sacrificed by decapitation 56 days after the induction of diabetes.The eyes were enucleated and the neural retina layers were carefully peeled off and preserved.The total neural retinal proteins were extracted from the control and diabetic groups, respectively,and then subjected to two dimensional gel electrophoresis (2-DE).Some different proteins spots were identified by peptide mass fingerprinting (PMF) as well as by tandem mass spectrometric (MS/MS) measurements.Western blot and indirect immunofluorescence (IMF) were used to confirmed that alpha;A-crystallin protein expression was upregulated in diabetic retina.Results An average of （3122plusmn;37） spots in normal retinas and（2702plusmn;21）spots in diabetic group were found by 2-DE image analysis software; about 150 spots in 2-DE gel of diabetic retinae exhibited statistically significant variations (t＞2.77,P＜0.05).Compared with normal rats' retinae, diabetic ones presented 68 protein spots of up regulation expression and 82 of downregulation expression in 2DE gel.Furthermore,20 of the 150 protein spots were identified by mass spectrometry.The points of 2369 and 1048 in 2-DE gel, showing high expression in diabetic retinal tissues, were identified as alpha;A-crystallin via PMF.Western blot validated that the expression level of alpha;A-crystallin in diabetic neural retina was much higher than that in the control group. Significantly increased expression of alpha;A-crystallin in nuclear retina in diabetic group was also observed by IMF. Fluorescence was mainly seen in the retinal nuclear layer;alpha;A-crystallin aggregation was detected in the perinuclear region of neurons.Conclusion The expression of alpha;A-crystallin increases in neural retina of early T2DM rats.
PURPOSE:To approach the establishment of t be optimal method for determining the intracellular free Ca2+ concentration[Ca2+]i of dissociated newborn rabbit retina cells by using fluorescent indicator-Fura-2/AM. METHODS:Trypsin was employed to prepare the retlna cell suspensions which were then loaded with Fura-2/AM followed by fluorescence determination. RESULTS:The cellular viability rate of retina cell suspensiotls prepared by 0.05% trypsin 10 minutes at 37deg;C was over 90%. Loading the retina cell suspensions with Fura-2/AM 40 minutes at 37deg;C and then measurlng the fluorescent intensity of the suspensions within 30 minutes were proved to be the optimum. CONCLUSIONS:The resting [Ca2+]i of retina cell suspension was (223plusmn;27)nmol/L whlch was within the expected range of [Ca2+]i level. 25mmoI/L and S0mmol/L K+ increased the [Ca2+Ji 59% and 148% respectively. These results indicate that the preparation of retina cell suspensions and the method of [Ca2+Ji determination are reliable and feasible. （Chin J Ocul Fundus Dis,1996,12: 108-110 ）